The envelope glycoprotein (Env) of HIV is a bipartite protein consisting of a surface subunit (gp120) that has receptor binding activity and a non-covalently associated transmembrane subunit (gp41) that has membrane fusion activity. Gp120 binding to cellular receptors triggers conformational changes that activate the fusion activity of gp41, which mediates delivery of the viral genome across the cell membrane. Our goals for this project are to elucidate structure-function relationships in the envelope glycoprotein in order to provide information for the rational design of novel HIV inhibitors and vaccines. We have undertaken extensive mutagenesis studies of a highly conserved coiled domain in gp41 that has been proposed to be a good target for the development of HIV inhibitors and vaccines. Previously we found that only a single mutation was tolerated among sixteen tested, indicating that this region is critical for activity and that drugs or antibodies directed to this region are unlikely to generate resistant mutants (Weng and Weiss 1998 J. Virol. 72:9676-9682). However, follow-up studies involving more extensive mutagenesis revealed two positions that tolerated multiple mutations, including residue 577 that lies at the edge of a hydrophobic pocket of the coiled coil that has been proposed to be a good target for drug development (Weng et al. 2000 J. Virol. 74:5368-5372.) These findings indicate that this region may be more susceptible to escape from inhibition, but our data also suggest that such mutants would be impaired in function. Furthermore, molecular modeling of the mutants shows that the function of the mutants can be readily explained by the effects of the mutations on the stability of the six-helix bundle, providing strong evidence for the importance of this structure in mediating membrane fusion. We are undertaking further mutagenesis to assess contributions of residues in the C helical domain of gp41. This information contributes to a foundation for the rational design of novel inhibitors and vaccines targeting the gp41.